Fluorescence and time-resolved phosphorescence plate readers are commonly used in time-resolved spectroscopy when evaluating dynamic processes in organisms, materials, or chemical compounds by means of spectroscopic techniques. Plate readers detect biological, chemical, or physical changes in samples contained within microtiter plates, and the measurements are usually based on the optical phenomena of absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescent polarization.
Although plate reader systems, such as those used with enzyme-linked immunosorbent assay (ELISA) reader, may efficiently and accurately measure biological parameters of a sample, they have associated disadvantages. Also, most platereaders do not work well with very small wells, for example, of less than 500-micron diameter. For example, diseases and conditions related to mitochondria dysfunction, such as muscle weakness, exercise intolerance, and amytrophic lateral sclerosis, may become detectable by measuring in a sample the rate of cellular oxygen consumption. However, the larger well sizes of standard microtitre plates (which are often as large as 100 to 200 microliters) fail to provide the necessary sensitivity to measure single-cell oxygen consumption. Finally, typical methods for measuring oxygen consumption with very small samples use charge coupled device (CCD) cameras for detecting phosphorescence, which can contribute significantly to the cost of the system. Not only are CCD cameras costly, but they can also be bulky and have a limited effective dynamic range (typically 8-10 bits).
The inverted microscope is relatively inexpensive and is a staple piece of laboratory equipment for observing living cells or organisms more easily than on a glass slide (as is used with a conventional microscope). However, an inverted microscope is not capable of taking the biological activity measurements of a plate reader, such as time-resolved phosphorescence measurements or very high accuracy quantitation of fluorescence changes with time. Both low cost, biological assessment capabilities, and traditional imaging is therefore desired.
Another medical tool for imaging and measuring biological parameters is fluorescence bronchoscopy. This technique is based on the principal that normal tissue fluoresces differently than diseased tissue when exposed to certain wavelengths of light. Many bronchoscopes use an ordered fiber optic array to detect this fluorescence and locate areas of damaged tissue. One problem with relying on fluorescence, however, is that body tissue may contribute a significant amount of background fluorescence noise. Therefore, an alternative and more accurate method of imaging diseased tissue is desired.
Provided herein is a device and system that not only expands the capabilities of a traditional inverted microscope by adding time-resolved plate-reader functionality, but also improves accuracy of the measurement of sample phosphorescence, including biological tissue in vivo. The device, which may be sized to fit into the condenser holder of an inverted microscope, provides plate-reading function at very low cost compared to that of traditional plate reader units, and also allows for the correlation of plate reader measurements with actual images of a specimen. Additionally, the device provides a much higher x-y spatial resolution than typical plate readers, and so allows for more accurate measurements from very small microwells, even as small as 100 microns in diameter or less. This feature is useful in high throughput screening, and the very small wells also enable more accurate measurements of cellular oxygen consumption even at the single cell level. Finally, the device and system also are able to measure other parameters associated with cell metabolism, such as pH changes, calcium uptake, and metabolic reduction of fluorescent dyes such as resorufin, in addition to and oxygen levels.